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Transforming variants of the avian myc-containing retrovirus FH3 arise prior to phenotypic selection
1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104-2092.
Abstract:
The avian retrovirus FH3, which encodes a Gag-Myc fusion protein, transforms chicken macrophages but not fibroblasts. However, passage of FH3 viral stock in fibroblasts leads to emergence of a virus capable of fibroblast transformation. This virus has not acquired myc mutations; instead, it carries internal gag deletions which confer the ability to transform fibroblasts. We now demonstrate that this and similar deletion variants emerge repeatedly during selection. Sequence analysis reveals direct repeats at or near deletion junctions, suggesting that errors during reverse transcription may be involved in genesis of these viruses, which are then positively selected in fibroblast culture. By using the polymerase chain reaction, we found that such variants preexisted in original stocks even before selection, although they could not be detected by focus assay.
Insights
Avian retrovirus FH3 gains fibroblast transforming ability through internal gag deletions, not myc mutations. These deletion variants repeatedly emerge and preexist in viral stocks, suggesting reverse transcription errors and positive selection.
Area of Science:
- * Molecular biology
- * Virology
- * Oncology
Background:
- * The avian retrovirus FH3 encodes a Gag-Myc fusion protein and transforms chicken macrophages but not fibroblasts.
- * Passaging FH3 viral stock in fibroblasts results in the emergence of a fibroblast-transforming virus.
- * This emergent virus transforms fibroblasts via internal gag deletions, not myc mutations.
Purpose of the Study:
- * To investigate the mechanism of fibroblast transformation by avian retrovirus FH3 variants.
- * To determine the origin and selection pressures of gag deletion variants.
- * To analyze the genetic basis for enhanced fibroblast transforming ability.
Main Methods:
- * Viral stock passage in fibroblast cell cultures.
- * Sequence analysis of emergent viral genomes.
- * Polymerase chain reaction (PCR) to detect pre-existing variants.
Main Results:
- * Internal gag deletions, not myc mutations, confer fibroblast transforming ability.
- * Deletion variants emerge repeatedly during selection in fibroblasts.
- * Sequence analysis indicates direct repeats at deletion junctions, suggesting reverse transcription errors.
- * PCR detected pre-existing deletion variants in original stocks before selection.
Conclusions:
- * Internal gag deletions are key to fibroblast transformation by FH3 variants.
- * Reverse transcription errors during viral replication likely generate these deletions.
- * Positive selection in fibroblast cultures favors the emergence of these transforming variants.
- * Pre-existing, low-level variants can be amplified under selective pressure.